Enduracidin derivatives

ABSTRACT

Enduracidin derivatives which have anti-microbial activity are produced by catalytic reduction of Enduracidin or a salt thereof.

United States Patent Miyake et al.

[54] ENDURACIDIN DERIVATIVES [21], Appl. No.: 778,300

[30] Foreign Application Priority Data Nov. 22, 1967 Japan ..42/7524 [52] US. Cl ..424/ll8, 424/123 [51] Int. Cl. ..A6lk 21/00 [58] Field ofSearch ..424/ll5l23 1 Sept. 26, 1972 [56] References Cited OTHER PUBLICATIONS Takeda, Del-went Farmdoc 37,713, NE 68, 16640, Pages 133- l4l, Published 5-27-69 Primary Examiner-Albert T. Meyers Assistant Examiner-Daren M. Stephens Attorney-Wenderoth, Lind & Ponack [57] ABSTRACT Enduracidin derivatives which have anti-microbial activity are produced by catalytic reduction of Enduracidin or a salt thereof.

4 Claims, No Drawings ENDURACIDIN DERIVATIVES DESCRIPTION OF INVENTION The present invention relates to new Enduracidin derivatives which are useful as antibiotics. More particularly, the present invention relates to reduction products of Enduracidin or its acid saltand processes for preparation thereof.

Enduracidin is the general name of an antibiotic which is produced by Streptomyces fungicidicus No; B- 5477 (ATCC-2 I013). The production and antibacterial properties are-reported-byE. Higashide era] (The Journal of Antibiotics An lntemational Journal Vol. XXI, No. 2, pp. 126437). The isolation and characterization of Enduracidin are reported by M. Asai et al (Ibid. Vol. XXI, No. 2, pp. 138-146) and the antimicrobial'activity of Enduracidin is reportedaby K. Tsuchiya et al (Ibid. Vol. XXI, No. 2, pp. 147-153). Enduracidin itself 'and the process for the preparation thereof are also patented in French Pat. No. 1,514,139, Belgian Pat. No. 688207, Spanish Pat. No. 332148, East German Pat. No. 58789, the Argentine Pat. No. 159523 and South African Pat. No. 66/6073, etc.

Enduracidin is an antibiotic of the peptide type which is composed of such moieties asaspartic acid, threonine, allothreonine, serine, glycine, alanine, ornithine, citrulline, a-amino-4-hydroxyphenylacetic acid,- a-amino-3,5-dichloro-4-hydroxyphenylacetic acid, fatty acid and two basic amino acids and others. This antibiotic shows the maximum absorptions in the infrared spectrum measured in 90 percent methanol at about 230 my. and 263mg It is a colorless powder which decomposes at 225 to 240 C and has very 'strong' antimicrobial activity against Gram-positive bacteria and phytopathogenic bacteria, andshows no cross-resistance with such known antibiotics as tetracycline, chloramphenicol, erythromycin, cephaloridine, oleandmycin, kanamycin, streptomycin, penicillin G, aminobenzyl-penicillin andneomycin.

It has been discovered that, in comparison with Enduracidin as such,reduction product has a considerably reduced toxicity, i.e. LD of Enduracidin reduction product is 350 mg/Kg. (intravenous injection in mice), while LD of Enduracidin itself is 33.5-60 mg/Kg..;in thesame condition. Further, Enduracidin reduction product has an improved stability and a therapeutic activity which is equally or more effective than Enduracidin itself.

The principal object of the present invention is to provide reduction products of Enduracidin or acid salt thereof which have lower toxicity to mammals and keep higher concentrations of the antibiotics in the blood against the hitherto-known Enduracidin.

Another object of this invention is to provide Enduracidin derivatives which have strong antimicrobial activity against Gram-positive bacteria and also even against such bacteria that have acquired resistanceto hitherto-known antibiotics such as streptomycin and neornycin.

'A'further object is to provide processes for preparing the novel and useful reduction products.

Still another object of the present invention is to provide new pharmaceuticalcompositions containing one or more of the reduction products.

Other objects of the present invention and advantageous features thereof will become apparent as the description proceeds.

Enduracidin reduction product is obtained by catalytic reduction of Enduracidin or a salt thereof in the presence of a metal catalyst at atmospheric or elevated pressure involving the use of, e.g. platinum, palladium.

Generally speaking, the reaction is carried out in a solvent which is non-reactive with Enduracidin or its salt and in which Enduracidin or its salt can be dissolved or suspended. Such a solvent is exemplified by water, methanol, acetic acid, and dioxane or a mixture thereof. As the starting material of this invention, of the reduction process, Enduracidin or a salt thereof may be used. As the salt of Enduracidin, there may be employed an organic salt or an inorganic salt. Examples of the inorganic salt include the hydrochloride, nitrate and the like, while said organic salt may be the formate, acetate, propionate, aspartate or glutamate and the like.

Said metal catalyst to be employed in the reaction, may be one which is generally used for catalytic reduction. For example, platinum, palladium, nickel or other catalysts possessing varied degrees of activity which catalysts can be used either as such or in the form supported by carriers. As for the amount of said catalyst, it is sufficient that the catalyst be present in amount from I to 10 percent based on the weight of Enduracidin. While the reaction pressure may be atmospheric, the reaction time is reduced when the reaction is conducted at an elevated pressure of the order of 20 KgJcm The reaction time ranges from 8 to 10 hours at atmospheric pressure and 4 to 8 hours at the elevated pressure. The time when the hydrogen ceased to be adsorbed may be regarded as the end point of the reaction.

Enduracidin reduction product thus obtained, may be used formedical purposes after proper purification or may be used for further reaction, as desired, directly or after the separation of the product from the reaction mixture.

In these reduction processes, about two moles of hydrogen gas per mole of Enduracidin or salt are absorbed. This fact supposes that two double bonds of Enduracidin are saturated by the catalytic reduction.

To separate the product from the reaction mixture, the catalystis, for example, filtered off and the filtrate is concentrated to dryness under reduced pressure. Then, the residue is washed with acetone or other organic solvent, and vacuum-dried in a desiccator. The resulting powder is sufficiently pure, but it may be further allowedto recrystallize from e.g. a mixture of methanol, acetone and water. In case a salt of Enduracidin has been reduced, the desired reduction product in free form may be obtained, for example, by a process where the reduction product is dissolved in a solvent, and the solution is passed columnwise over weakly basic ion exchange resins, or neutralized to pH 8 to 8.5 with a tertiary aminesuch as trialkylamine and, if necessary, a solvent in which the product is sparingly soluble is added so as to cause precipitation oflthe product.

The reduction products of Enduracidin or acid salts are useful as antibiotic agents and generally administered in the form of powder, capsule, syrup, oil, injection, ointment, tablet, etc., i.e. orally, parenterally, topically or externally. Pharmaceutical compositions containing one or more of the reduction products can be prepared according to any per se conventional means for the preparation of capsules, syrups, oils, injections, etc.

ln the aforesaid various administrational forms, the active ingredient may be present in a minor proportion with the carrier constituting the major proportion. However, the reverse relationship may also be possible, so that a minor proportion of carrier is employed in association with a major proportion of active ingredient.

Examples of administrational compositions are hereinafter exemplified.

Ointment Enduracidin reduction product I g.

Methyl para-hydroxybenzoate 0.12 g.

Propyl para-hydroxybenzoate 0.03 g.

Anhydrous lanoline 9.85 g.

White petrolatum 89 g.

The ointment is used for the treatment of superficial cutaneous and mucosal infections.

Solution Enduracidin reduction product 0.5 g. Distilled water to make l ml. The solution is administered for the treatment of the suppurative middle ear atitis.

Oils

Enduracidin reduction product 0.5 g.

Cera alba 4.8 g.

Peanut oil to make 100 ml.

Injection Enduracidin reduction product 25 mg.

Polyoxyethylene ether of hydrogenated castor oil 50 mg.

lN-hydrochloride 0.057 ml.

Sterilized distilled Water to make l ml.

Tablets Enduracidin reduction product 0.l g.

Lactose 0. l95 g.

Cornmeal 0.l g.

Magnesium stearate 0.005 g.

Total: 0.4 gflablet The pharmaceutical compositions show a greater therapeutic activity against infections caused by Streptococcus pyogenes, Staphylococcus aureus, Diplococcus pneumoniae, etc. than that of hitherto known antibiotics.

A typical effective daily dose of the reduction product is usually about 0.02 to mg/KgJday, desirably 0.1 to 0.5 mg/Kg., for injection, although an increased or reduced daily dose is also effective depending on the severity of the infection being treated.

The following examples show presently preferred embodiments of this invention. It is to be understood that the following examples are solely for the purpose of illustration and not for limitation of this invention, and that variations may be resorted to without departing from the spirit of the invention. In the examples, parts by weight bear the same relation to parts by volume as do grams to milliliters.

EXAMPLE 1 In 100 parts by volume of 80 percent acetic acid is suspended 2 parts by weight of platinum oxide, and the suspension is thoroughly stirred in hydrogen gas streams so that the hydrogen gas is absorbed thereto. On the other hand, 20 parts by weight of Enduracidin hydrochloride is dissolved in 300 parts by volume of 80 percent acetic acid, and the resulting solution is added to the above suspension. The mixture is vigorously stirred in hydrogen gas stream at atmospheric pressure until the hydrogen gas ceases to be absorbed (about 385 parts by volume of hydrogen gas is absorbed). The reaction mixture is subjected to the filtration of the catalyst and the filtrate is condensed under reduced pressure. The residue is washed with acetone several times and, then, vacuum-dried in a desiccator packed with phosphorus pentoxide to obtain colorless powder of the Enduracidin hydrochloride reduction product.

Yield: 1.8 parts by weight. 1. Decomposition point 234-240 C (Enduracidin hydrochloride 235245 C) 2. Elementary analysis:

Reduction Product Hydro chloride Solvents Rf value Acetic acid n-Butanol. Water (124:5) 0.45 z 0.] n-Butanol saturated with water 0.0 Pyridine. n-Butanol. Water (324:7) 0.80 I 0.]

These values are the same as those of hydrochloride.

original Enduracidin 6. Color Reaction:

Reagent Result Ninhydrin reagent positive (palebrownish violet) Barton's reagent (a mixture of equal volume of l% aqueous ferric positive chloride and l% aqueous potassium (pale-blue) ferricyanate) Dragendorff reagent positive (orangish-yellow) Alkaline potassium permanganate discoloration occurs (reduction) These results are the same as those of original Enduracidin hydrochloride.

7. Ultraviolet absorption spectrum:

The significant maximum-absorptions observed are as follows:

Reduction Product Enduracidin hydrochloride 230 m, (g m =l) 230 m u 10 NC! nnex" 276 mp. (q =32.4)

213 mu G 287 mu (e l2) (shoulder) 8. Infrared absorption spectrum:

The infrared absorption spectrum is observed by the potassium bromide disk method. The significant absorption bands in microns are listed in the following table and the results are the same as those of original Enduracidin itself.

3.05 (strong) 3.25 (shoulder) 3.38 (shoulder) 3.45 (strong) 5.75 (shoulder) 5.95 (shoulder) 6.09 (strong) 6.23 (shoulder) 6.47 (shoulder) 6.55 (shoulder) 6.61 (strong) 6.95 (shoulder) 7.25 (middle) 8. l 6 (broad, strong) 8.51 (middle) 9.89 (middle) 10.40 (weak) 11.93 (middle) 12.30 (weak) 9. Toxicity The toxicity of the product as-determined withmice is LD =350 mg'JKg. (intravenous injection) orabout l-g./Kg. (intraperitoneal injection) in mice. On the other hand, LD of original Enduracidin in mice is 33.5-60 mgJKg. (intravenous injection).

10. Antibacterial spectra: t

The antibacterial spectrum of the product as measured on TSA medium enriched with cattle blood is as follows:

Minimum inhibitory concentratiOMmcymI) reduction Original Test microorganism The result of a therapeutic test in mice infected with Staphylococcus aureus 308 A-l is as follows.

Surviving animals, in heads Survival rate animals tested, in heads dosage(mg./kg.)

100 50 25- 12.5 6.2 3.l ED Amount of (mg/kg.) inoculum (LDM 5.6 5/5 5/5 5/5 4/5 4/5 US 4.83 dosage(mg./kg.)

40 20 10 5 5D,,, (mg/kg.) Amount of inoculum an) .A multiple of Li) Thus, the ED of this product is 5.0 mgJKg. when the amount of inoculation is 5, 6 times the median lethal dose LD The ED of Enduracidin under similar condition is 8.0 mgJKg.

EXAMPLE 2 17 parts by weight of the reduction product of Enduracidin acetate as colorless powder is obtained in the same manner as in Example 1 except employing Enduracidin acetate instead of Enduracidin hydrochloride.

Decomposition point: 232-240" C.

Infrared absorption 3.06, 3.45, 5.75(shoulder), 5.9(sh0ulder),

6.1, 6.45(shoulder), 6.7, 6.9, 7.3, 7.9-8.1,

Ultraviolet absorption A max 230 mp. (e 199, in methanol) A max 276 mp. (e 32.2, in 90%, methanol) EXAMPLE 3 [n 50 ml. of 70 percent methanol is dissolved 10 g. of Enduracidin hydrochloride reduction product prepared in Example I, while heating at 50 C. With vigorous stirring, triethylamine is added dropwise to the solution so as to adjust the latter of pH 8.0 8.5.

The solution is allowed to stand overnight in a refrigerator and the resulting precipitates are separated centrifugally and washed twice with water. Then these precipitates are vacuum-dried in a desiccator, 8.8 g. of a colorless powdery reduction product in free form are obtained.

Decomposition point: 238 245 C.

Elementary analysis:

[01],, 53.2 (C=0.5, dimethylforrnamide).

This product is sparingly soluble in water relative to Enduracidin hydrochloride reduction product.

Infrared absorption 3.07, 3.45, 5.75(shoulder), 5.9(shoulder),

6.1, 6.45(shoulder), 6.6, 6.8, 7.2, 7.9-8. 1

Ultraviolet absorption A max 264 mu (e 54.6, in methanol) A max 310 mu (s 17.7, in 95% methanol) A max 230 mu (e 201, in N/lO HCl) A max 276 mu (e 32.4, in-N/lO HCl) A max 282 m .t(e 30.9, in N/lO HCl) A max 250 my. (e,,,,,,'* 289, in N/lO NaOl-l) A max 293 mp. (6 68, in N/lO NaOH) EXAMPLE 4 25 parts by weight of Enduracidin hydrochloride is dissolved in 250 parts by volume of 70% methanol, followed by the dissolution of 6 parts by volume of acetic acid. 50 parts by weight of 5 percent palladium-activated carbon (50 percent moisture) is added to the lo above solution so that the reduction takes place at an initial pressure of KgJcm of hydrogen gas and at room temperature, continuing until no more hydrogen gas is absorbed (3.5 hours). The catalyst is centrifugally discarded and the reaction mixture is subjected to the filtration using Toyo Roshi No. 5 C (Toyo Roshi Kaisha Ltd.) as a filter paper. The filtrate is concentrated to dryness under reduced pressure and washed with acetone. The product is then vacuum-dried in a desiccator, whereupon 19.4 parts by weight of a colorless powdery reduction product in hydrochloride form is obtained.

Decomposition point 230-233 C.

Elementary analysis:

lnfrared absorption 3.06, 3.45, 5.75(shoulder), 5.9(shoulder) 6.1, 6.45(shoulder), 6.6, 6.9, 7.2, 7.9-8.1,

Ultraviolet absorption A max 230 1'l1[L( 200, in 90% methanol) A max 275 my (6 31.5, in 90% methanol) EXAMPLE 5 parts by weight of Enduracidin hydrochloride is dissolved in 250 parts by volume of 70 percent methanol, followed by addition of 6 parts by volume of acetic acid. To the solution is added a platinum black, which is previously prepared from 0.5 part by weight of platinum oxide, so that the reduction takes place at an initial pressure of 20 l(g./cm of hydrogen gas and at room temperature for 4 hours. Then, the catalyst is filtered off and the filtrate is concentrated to dryness under reduced pressure. The residue is washed with acetone and vacuum-dried in a desiccator. The above procedure yields 22.2 parts by weight of a colorless powdery Enduracidin .reduction product in hydrochloride form.

Decomposition point: 230233 C.

Elementary analysis:

Concentrations of antibiotics (y/ml. in plasma) in the blood on intramuscular injection into rats (6.25 mg./Kg.).

Time, in hours after injection 2 4 8 12 24 Antibiotics Enduracidin 4.0.5 5.6 6.5 Enduracidin reduction 8t-5 6.3 8.6 5.5

product Concentrations of antibiotics (ylml. in plasma) in blood on intramuscular injection into rabbits (8 mgJKB.)

Hours after injection Antibiotics 1 3 6 24 Enduracidin 2.75 3.20 4.30 2.45 Enduracidin reduction 6.27 7.51 6.81 5.38

product What is claimed is:

1. Reduction product of Enduracidin or its acid salt wherein the properties of the hydrochloride is as follows:

1. its decomposition point is 234-240 C,

2. its specific rotation is [a],," 82.8 (C=l.0,

percent methanol), 3. Rf values obtained in paper partition ehromatography are as follows:

Solvents Rf value Acetic acid: n-Butanol: 0.45 1 0.1

Water 1:4:5) n-Butanol saturated with 0.0 water Pyridine: n-Butanol:

0.80 z 0. 1 Water (324:7)

4. its significant maximum-absorptions in ultraviolet spectrum are as follows:

A,,,,,,""" HCl= 230 my. (c 201) 276 my. (s 32.4) 282 mp. (e 30.9) 250 mp. 6 289) 293 i 68) 6. its solubilities are as follows: Slightly soluble in water; soluble in aqueous methanol, aqueous acetone, aqueous acetic acid, dimethylformamide, pyridine and dioxane;

insoluble in acetone, ethyl acetate, ether,

chloroform.

7. its color reactions are as follows:

Reagent Result 10. it shows antimicrobial activity against gram-posi- Ninhydrin reagent positive (palebrownish violet) Barton's reagent (a mixture of equal volume of 1% aqueous ferric chloride and 1% aqueous potassium ferricyanate) Dragenorfi reagent positive (pale-blue) positive (orangishyellow) discoloration occurs (reduction) Alkaline potassium permanganate 8. its elementary analysis is C 52.5 i 0.5, H 6.22

$0.3, N= 14.21 $0.3 and Cl=4.5l 20.3.

9. it's LD value in mice is 350 mg/Kg. (intravenous 3. The Enduracidin reduction product as claimed in claim 2, in the form of the hydrochloride.

4. The Enduracidin reduction product as claimed in claim 2, in the form of the acetate.

injection) and l g/Kg. (intraperitoneal injection). 1 

2. its specific rotation is ( Alpha )D26 + 82.8* (C 1.0, 70 percent methanol),
 2. The compound as claimed in claim 1, wherein the acid is selected from the group consisting of formic, acetic, propionic, aspartic, glutamic, hydrochloric and nitric acids.
 3. The Enduracidin reduction product as claimed in claim 2, in the form of the hydrochloride.
 3. Rf values obtained in paper partition chromatography are as follows: Solvents Rf value Acetic acid: n-Butanol: 0.45 + or - 0.1 Water (1:4:5) n-Butanol saturated with 0.0 water Pyridine: n-Butanol: 0.80 + or - 0.1 Water (3:4:7)
 4. its significant maximum-absorptions in ultraviolet spectrum are as foLlows: lambda maxN/10 HCl 230 m Mu ( epsilon 1cm1% 201) 276 m Mu ( epsilon 1cm1% 32.4) 282 m Mu ( epsilon 1cm1% 30.9) lambda maxN/10 NaOh 250 m Mu ( epsilon 1cm1% 289) 293 m Mu ( epsilon 1cm1% 68)
 4. The Enduracidin reduction product as claimed in claim 2, in the form of the acetate.
 5. its significant absorptions in Infrared spectrum (potassium bromide dish) are as follows: 3.02 (strong) 3.25 (shoulder) 3.38 (shoulder) 3.45 (strong) 5.75 (shoulder) 5.95 (shoulder) 6.09 (strong) 6.23 (shoulder) 6.47 (shoulder) 6.55 (shoulder) 6.61 (strong) 6.95 (shoulder) 7.25 (middle) 8.16 (broad, strong) 8.51 (middle) 9.89 (middle) 10.40 (weak) 11.93 (middle) 12.30 (weak)
 6. its solubilities are as follows: Slightly soluble in water; soluble in aqueous methanol, aqueous acetone, aqueous acetic acid, dimethylformamide, pyridine and dioxane; insoluble in acetone, ethyl acetate, ether, chloroform.
 7. its color reactions are as follows: Reagent Result Ninhydrin reagent positive (pale-brownish violet) Barton''s reagent (a mixture of equal volume of 1% aqueous ferric positive (pale-blue) chloride and 1% aqueous potassium ferricyanate) Dragenorff reagent positive (orangish-yellow) Alkaline potassium discoloration permanganate occurs (reduction)
 8. its elementary analysis is C 52.5 + or - 0.5, H 6.22 + or - 0.3, N 14.21 + or - 0.3 and Cl 4.51 + or - 0.3.
 9. its LD50 value in mice is 350 mg/Kg. (intravenous injection) and 1 g/Kg. (intraperitoneal injection).
 10. it shows antimicrobial activity against gram-positive bacteria and phytopathogenic bacteria and no cross-resistance with tetracycline, chloramphenicol, erythromycin, cephaloridine, oleandmycin, kanamycin, streptomycin, penicillin-G, aminobenzyl-penicillin and neomycin. 